Biosynthesis and characterization of zinc oxide nanoparticles using Nigella sativa against coccidiosis in commercial poultry

Coccidiosis causes huge economic losses worldwide. Current study evaluated the effect of biosynthesized Zinc oxide nanoparticles (ZnONPs) using Nigella sativa, on Eimeria tenella infected broilers. Scanning electron microscopy showed spherical ZnONPs with 50–100 nm diameter, Fourier transforms infrared spectroscopy revealed the functional groups involved in the reduction of zinc acetate dihydrate to ZnONPs, UV–vis spectroscopy showed a peak at 354 nm, and Zeta potential exhibited stability at − 30 mV. A total of 150, a day-old broiler chicks were divided into 5 equal groups. Control negative: uninfected and untreated; Control positive: Infected and untreated; 3rd, 4th and 5th group were infected orally with 5 × 104 sporulated oocysts of Eimeria tenella and treated with 60 mg/kg ZnONPs, 1% Nigella sativa seeds and amprolium 125 ppm, respectively. ZnONPs significantly (p < 0.05) improved the growth performance in the infected birds and decreased the oocyst shedding and anti-coccidial index. A significant (p < 0.05) decrease in the level of aspartate transferase and alanine transferase, whereas, a significantly higher amount of antioxidants like catalase and superoxide dismutase in ZnONPs treated group was observed. Pro-inflammatory cytokines like IL-2 and TNF-α were significantly decreased by ZnONPs (p < 0.05). In conclusion, biogenic ZnONPs with Nigella sativa might have enhanced anticoccidial, antioxidant, and anti-inflammatory effects with improved growth performance.

Green synthesis of zinc oxide nanoparticles. Zinc acetate dihydrate salt (0.25 M) solution was prepared by adding 22.92 g zinc acetate dihydrate salt to 500 ml of distilled water in a flask. Further, 10 ml of Nigella sativa seed extract added dropwise into the zinc acetate solution under continuous stirring at 60 ℃ for 2 h on a magnetic stirrer. While stirring, NaOH was added dropwise in the above solution to maintain the pH at 12. Plant extract acted as a reducing agent. The formation of light yellow color suspended particles and visible color change of the solution from brown to yellow indicated the formation of zinc oxide nanoparticles. This mixture was then centrifuged at 6000 × g for 15 min and supernatant was discarded to collect the pellet in a Petri plate. Afterwards, it was placed overnight in a hot air oven for drying at 60 ℃. Calcination of yellow-colored ZnONPs was done in a muffle furnace at 400 ℃ for 4 h to remove impurities (Figs. 1,2) 30 .
Influence of the plant extract on production of ZnONPs was investigated by varying the ratio of plant extract to salt (v/w) and yield of prepared ZnONPs was calculated by following formula Characterization of zinc oxide nanoparticles. The optical properties of Zinc oxide nanoparticles (ZnONPs) were analyzed by using a UV-spectrophotometer. Firstly, water was run as a blank, and then the ZnONPs sample was set in a spectrophotometer. UV light passed at 354 nm and confirmed the formation of ZnONPs 31 . Scanning Electron Microscopy was used to identify the morphology of ZnONPs. The sample was observed at 20kv with a frequency of 2838cps. For determination of shape and size, ZnONPs was analyzed at different resolution and magnification power. Fourier transform infrared spectroscopy (FTIR) analysis was performed to know the functional groups involved in the synthesis of ZnONPs. The solution was dried at 75˚C and characterization was done at a wavenumber ranging from 4000 to 400 cm −129 . Stability of green synthesized ZnONPs were analyzed by Zeta potential 32 . To determine the purity and structural characteristics of ZnONPs, diffraction intensities was recorded at 30 kV and 10 mA current with the range 2θ from − 3 to 160°by using X-ray diffractometer (Bruker D2 Phaser). Feces

Experimental design
The experiments were performed in accordance with comprehensive animal welfare guide (FASS, 2010) and approved by the Ethical Review Committee (ERC) of the University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan (permit No. DR/ 357). This study also followed the ARRIVE guidelines. A total of 150, day-old broiler chicks were purchased from a nearby hatchery and kept under standard conditions. Birds were divided into five equal groups. On the 28th day except for group 1, all groups were infected orally through a stomach tube, with 5 × 10 4 sporulated oocysts of Eimeria tenella. Group 1 was control negative, which was kept un-infected and un-medicated. Group 2 was control positive, infected, and un-medicated. Group 3 was fed with 60 mg/kg ZnONPs orally through a stomach tube. Group 4 was fed with 1% Nigella sativa crushed seeds mixed in feed. Group 5 was given amprolium at a dose rate of 125 ppm orally. Treatment was given from 28 to 35th day of trial.
Anticoccidial activity analysis. Anticoccidial activity of ZnONPs was analyzed by recording the body weight gain (BWG), FCR, OPG, mortality rate, lesion scores, and anticoccidial index of the experimentally infected birds.
Determination of weight gain and feed conversion ratio (FCR). Growth performance, body weight, and feed intake of the birds were recorded on weekly basis 34 . Mortality rate and lesion score. Birds were observed daily for mortality. At the end of the trial, birds from each group were slaughtered and examined for cecal lesions, and scored on the basisof severity of lesions. 0 score for -ve group and 4 for severely affected and dead birds 36 .
Anticoccidial index (ACI). Anticoccidial index of each group was calculated by the formula: (Relative ratio of the body weight gain + survival rate)-(lesion index + oocyst value). Anti-coccidial index greater than 180 is perceived to have a very effective anticoccidial action. ACI in 160-180 range indicated marked anticoccidial action and ACI less than 120 indicated no anticoccidial activity 37 .
Blood sampling and serum biochemical analysis. Blood samples (2 ml) were collected on 35th day from the wing vein of all the birds in plain tubes, allowed to clot and then centrifuged at 4000 rpm for 10 min. The obtained serum was stored at − 20 °C for biochemical analysis 38 . Serum biochemical profile including aspartate transferase (AST), and alanine transferase (ALT) was evaluated by using a Diasys kit following the manufacturer's guidelines 39 . Determination of antioxidant enzymes activity. Antioxidants like catalase (CAT) and superoxide dismutase (SOD) activities were evaluated by using Fine test Elisa kits following guidelines given by the manufacturer 40 .

Determination of serum inflammatory cytokines. The determination of tumor necrosis factor-alpha
(TNF-α) was evaluated by using the BTlab ELISA kit and level of Interleukins-2 (IL-2) was determined by using a Fine test ELISA kit following the manufacturer's guidelines 41 .
Statistical analysis. The data were analyzed by using one-way ANOVA using SPSS version 20.0 and difference between mean was determined using Tukey's test at (p < 0.05) level.

Ultra violet analysis of zinc oxide nanoparticles. Ultraviolet spectroscopy confirmed the synthesis of
ZnONPs and showed the optical activity and stability of nanoparticles. After the formation of yellow suspended particles, this solution was run in an ultraviolet spectrophotometer. The light passed and spectra observed at 354 nm indicated ZnONPs as shown in (Fig. 3).
Zeta potential. Results of zeta potential at − 30 mV indicated the colloidal stability of green synthesized ZNONPs (Fig. 7).
Growth performance. An increase in body weight was noted in birds of all groups and there was no significant difference (p > 0.05) among different groups till the onset of clinical signs of the coccidiosis. Growth performance decreased significantly (p < 0.05) due to Eimeria infection. Treatment with ZnONPs and Nigella sativa significantly (p > 0.05) enhanced the growth performance and the results were comparable to that of the amprolium-treated group (Table 1).

Oocysts shedding and lesion scores.
On the 4th day post-infection, there was frequent bloody diarrhea in the infected groups and generalized weakness was present in the birds. No. of oocysts per gram (OPG) of feces was counted by McMaster's chamber on, 32th, 33th, 34th, and 35th days. On the 4th day post-infection, a large number of oocysts was shedding in the feces and maximum OPG was found in the control positive group, which was significantly higher (p < 0.05) than in other groups. The non-infected group was free from any signs    www.nature.com/scientificreports/ and oocyst shedding. In treatment groups, OPG of ZnONPs groups was lower than in NS seeds and amprolium treated groups. Lesion score was significantly higher in control + ve group indicating the cecal damage. Lesion scores in ZnONPs were significantly low (p < 0.05) and comparable to that of amprolium treated group (Table 2).

Anticoccidial index (ACI).
There was 16% mortality in the control positive group and no mortality in the treated and control negative group. Lesion index was calculated by multiplying lesion score by 10. ACI of the control positive group was < 120 indicating no anticoccidial effect. ZnONPs treated group showed ACI > 180 comparable to that of amprolium treated group (p > 0.05) (Fig. 8).
Serum biochemical analysis. The current study observed an increased level of ALT and AST in the serum due to tissue damage 7 days post-infection (Fig. 8). Amount of ALT and AST showed a significant (p < 0.05) increase in an infected group as compared to the non-infected group. Group treated with ZnONPs has shown a significant effect (p < 0.05) on the amount of ALT and AST comparable to amprolium treated group.
Antioxidant enzyme activities. The current study observed a significant (p < 0.05) decrease in the amount of SOD and CAT in control positive group. The level of SOD and CAT was significantly increased www.nature.com/scientificreports/ (p < 0.05) in infected but treated groups. ZnONPs showed a significant effect (p < 0.05) as compared to other treatment groups (Fig. 8).

Serum inflammatory cytokines.
The amount of IL-2 and TNF-α was significantly higher (p < 0.05) in the infected group than control negative and other treated groups. ZnONPs and NS seed groups showed a significant decline in the level of IL-2 and TNF-α. The level of IL-2 and TNF-α in amprolium group was significantly lower (p < 0.05) than that of control positive group but higher than other groups (Fig. 9).

Discussion
Poultry coccidiosis is a very harmful disease that damages the gastrointestinal tract of the birds causing huge economic losses. Prevailing resistance against available anticoccidials provokes the use of alternative treatments for coccidiosis like natural products 42 . Moreover, the drugs used against coccidiosis like toltrazuril cause the problem of drug residues in poultry products i.e. meat and eggs 43 . That's why nanoparticles and herbal plants are being used as a new and effective remedy against coccidiosis with maximum efficacy and no residue problem 44 . In this study, anticoccidial effect of green synthesized ZnONPs was evaluated as compared to traditional zinc. During green synthesis, a color change from light brown to yellow was observed indicating the reduction of zinc 45 . Bioactive components like quinones, alkaloids, phenolics and flavonoids present in Nigella sativa extract reduced the zinc acetate salt and control the size of ZnONPs. Carboxylic and amino groups of these bioactive compounds binds to the surface of zinc (Zn +2 ) and amides from capping proteins may act as a stablizer 46 . Plant extract concentration is critical in the production of ZnO NPs. Hence, 3 ml/gm and 6 ml/gm don't have sufficient bioactive components that can reduce Zinc acetate and lead to low yield of NPs. Whereas, 10 ml/gm has given highest yield of ZnONPs 47 (Fig. 2). Confirmation of synthesis of ZnONPs was done by Uv-analysis. In Uv-spectrum, a clear absorption peak was formed at 354 nm, indicating the presence of ZnONPs. In another study by 48 , ZnONPs were synthesized by using Cassia fistula and Melia azedarach which revealed absorption peak at 320 nm and 324 nm, respectively 49 reported that ZnONPs synthesized by leaf extract of Piper betle showed absorption peaks at 358, 368, and 378 nm. This low absorbance is due to presence of complex biomolecules in the plants 50 .
Scanning Electron Microscopy images showed more or less spherical NPs of 50-100 nm size range 51 reported that the diameter of ZnONPs synthesized by Mentha Spicata extract was 11-80 nm. The difference in the size of the NPs is because of the difference in temperature conditions provided during the synthesis of NPs. If a high temperature is provided to the reaction mixture, small-sized NPs will be synthesized 52,53 . The volume of the plant extract also plays a role in controlling the size and shape of NPs. Using a large volume of extract during synthesis will reduce the size of NPs 54,55 . FTIR spectrum showed a peak at 3861 cm −1 and 3746 cm −1 representing the stretching vibration of hydroxyl OH-bond from phenolic compounds present in the plant 29 . The peak at 2367 cm −1 indicated N-H bond of the primary and secondary amides. The band at 1395 cm −1 indicated C=O stretching of amides and C=C stretching of alkenes 56 . The band at 1087 cm −1 corresponded with stretching of C-O bond 57,58 . The peaks at 850 cm −1 and 760 cm −1 indicated C-N stretch of the amine group 59 . The absorption peak at 490 cm −1 showed the ZnO bond as reported previously 60 . The presence of alkenes is confirmed by FTIR spectra that have a role in antioxidant activity 61 . Hydroxyl bonds confirmed the presence of flavonoids, having antioxidant and anti-inflammatory properties 62 and C=O groups indicateed the presence of thymoquinone responsible for anti-coccidial activity 63 . Zeta potential of − 30 mV showed good stability of green synthesized ZnONPs. Negative surface charge is due to the strong binding affinity of extract chemicals for the NPs which increased their stability and reduced their tendency to aggregate 64 . Distinct narrow peaks of XRD showed that ZnONPs are free from impurities and have hexagonal wurtzite crystals 47 .
In this study, a significant decrease in growth performance was observed in an infected group similar to the studies reported by 65 . The reduction in the growth performance may occur due to intestinal damage and decreased length of the villi which may have resulted in poor nutrient absorption 66 . Infected but treated groups, with ZnONPs, NS seeds, and amprolium respectively, showed improvement in growth performance because of decreased intestinal damage 36 reported that the supplementation of the Nigella sativa reduced the adverse www.nature.com/scientificreports/ effects of Eimeria and increased the growth performance. ZnONPs are better than the traditional Zn sources in improving feed utilization and growth because Zn is involved in many enzymatic reactions of metabolism 67 . ZnONPs promote growth because they increase the intestinal villi length and crypt depth and hence resulting in increased nutrient absorption 68 .
The results of this study showed that there was severe bloody diarrhea in the control positive group and a maximum number of oocysts shedding in the feces. Bloody diarrhea occurred due to penetration of spores inside the cecal epithelium and spores of protozoans complete their developmental stages inside the epithelium till the schizogony stage, ultimately damaging the intestinal blood vessels and causing hemorrhages 69,70 . Current study revealed that ZnONPs significantly decreased the number of E. tenella oocysts in the feces, caecum lesion score and anticoccidial index at a dose of 60 mg/kg similar to 71 . This may be due to the reason that ZnONPs diminished www.nature.com/scientificreports/ the growth and development of protozoa inside the intestinal cells before the inactive oocysts were formed and shed 44 . Furthermore, NS seeds given in diet also decreased the OPG in E.tenella-infected birds 36 . Current study showed a significant increase level of AST and ALT in the serum of infected birds. Similar results were reported by 10 and they proposed that this rise in enzyme activity occurs due to cecal cell wall damage, inflammation, and heavy blood loss. In this study, ZnONPs and NS seeds caused a reduction in the level of these enzymes. It is thought that quinone component present in NS seeds may have a role in reducing cecal lesions 72 . Our results are in line with the study of 36 which showed that the treatment with NS seeds reduced the level of these enzymes. In contrast 73 , reported that there was no significant change in the AST and ALT levels in infected and treated groups. Moreover 74 , stated that the level of ALT was enhanced after E. tenella infection, but there was no effect of infection on level of AST. This contradiction in results may occur because of variations in the degree of intestinal inflammation, intestinal damage and hemorrhages 75 .
Results of antioxidant activity showed a significant decrease in the amount of SOD and CAT, indicating oxidative damage because of Eimeria infection 76 . While the level of SOD and CAT was high in treatment groups similar to the findings of 77 who stated that Nigella sativa significantly increased the level of SOD and CAT. In our study, the antioxidant activity of ZnONPs was higher than the other treated groups similar to the results of 78,79 who stated that administering ZnONPs in the broiler diet significantly increases both SOD and CAT activity in serum. However, in contrast to our study, (El Katcha et al. 2017) reported that there was no significant change in antioxidant activity upon feeding 15-60 ppm nano-Zinc to the broilers. Moreover, studies have reported that 60 mg/kg ZnONPs have good Cu-Zn-SOD activity. The reason for the marked antioxidant property of ZnONPs is that Zn is an important constituent of SOD and this enzyme reduces oxidative stress by efficiently scavenging the superoxide free radicals 80 . Another mechanism behind the antioxidant activity of ZnONPs is the competition of zinc with iron and copper for attachment on the binding site present on cell membranes and that's why Zn decreases free radical production 81 . Moreover, it is also proposed that Zn promotes the production of metallothionein and this protein plays an important role in the detoxification of free radicals 82 and also activates the antioxidant proteins and enzymes like glutathione peroxidase (GPx) and CAT 83 .
This study indicated a significantly high level of pro-inflammatory cytokines in broilers of the control positive group 84 showed that in intestinal inflammation, pro-inflammatory cytokines intensely increased because these are attracted to the site of inflammation. Treatment with biogenic ZnONPs diminished the pro-inflammatory cytokines (IL-2 and TNF-α). Our results are in agreement with 85 , who reported that ZnONPs decreased the mRNA expression of IL-2 and TNF-α in serum. The anti-inflammatory property of Zn may be due to Nrf2/HO-1 signal pathway 86,87 . Substances that activates the Nrf2 pathway, decrease the overproduction of pro-inflammatory cytokines like IL-2 and TNF-α, and enhances immunoglobulin production 88,89,90,91 also stated that an activated Nrf2/HO-1pathway; stops the TLR4-mediated inflammatory responses. It is shown that ZnONPs have anticoccidial property and improved growth performance.

Conclusion
Biogenic ZnONPs supplementation to Eimeria tenella infected broilers showed anticoccidial activity by decreasing the oocysts shedding and improve the growth performance. Moreover, ZnONPs decreased the tissue damage which was indicated by decrease in the level of serum enzymes like alanine transferase and aspartate transferase. ZnONPs showed antioxidant and anti-inflammatory activity by significantly increasing the activity of superoxide dismutase alongwith catalase and decreasing the amount of pro-inflammatory cytokines interleukins-2 as well as tumor necrosis factor-α. So, ZnONPs can be used as an alternative to treat coccidiosis. But the exact mechanism of its anti-coccidial action is still unknown so further research is needed to understand its mechanism of action alongwith comparison between different plant mediated synthesis of ZnONPs.

Data availability
All data generated or analyzed during this study are included in this manuscript.